PMC

Functionality of Nup153FG hydrogels by fluorescence microscopy. (a) Laser-reflection image of a Nup153FG hydrogel allows positioning the gel before fluorescence image acquisition (inverted colors). (b) Selectivity of Nup153FG hydrogels. Spectrally separated confocal images of a 70 kDa dextran labeled with FITC and Importin β labeled with Alexa 594 were taken simultaneously. The hydrogel (located on the right side of the dashed line) shows Alexa 594 fluorescence (Importin β) in the hydrogel after 10 min, while FITC fluorescence (dextran) remains outside the hydrogel. (Lower panel) Corresponding fluorescence intensity analysis of FITC and Alexa 594 fluorescence. Scale bar is 10 μm.

Labeling design and AA composition of Nup153FG. (a) Alexa 594-maleimide (A) attached to a unique cysteine in red and Alexa 488-hydroxylamine (D) coupled to the site-specifically introduced unnatural amino acid with ketone functionality (in green). (b) Different classes of FG-repeats are color-coded and segments analyzed by single-molecule FRET are separated (dashed lines) and labeled α to ζ (upper row). (Middle row) Digital representation of AA charge. (Lower row) Analysis of N and Q content along the sequence with a sliding window of 10 AAs. AA composition was analyzed with the software EMBOSS ().

Comparison and classification of Nup153FG segments. (a) Dependence of R_H on molecular weight (segment length). The predicted curve for a relaxed behavior of a polypeptide chain (solid representation) (). The previously determined confidence interval for this relaxed coil behavior and thus the boundaries of collapsed and extended coil behavior are visualized by the shaded gradient. (b) Relation of R_E to % charge/% hydrophobicity (analog to Yamada et al. ()). Measured R_E were related to the end-to-end distance predicted according to the relaxed coil model (R_E,RC) to provide a segment-length-independent measure of distances. (Solid line) Position where a segment with ideal relaxed coil behavior would occur. (Shaded) Relaxed coil regions. (c) Dependence of R_E/R_E,RC from the content of N and Q within the probed segment. (Solid line) Linear fit through all data-points. (Solid circles) Distances measured under native conditions. (Open circles) Distances under unfolding conditions (4 M GdmCl).

MFD smFRET analysis of the Nup153FG segments γ and ε. (a) Burst-integrated fluorescence-lifetime analysis of segment ε (upper row) and segment γ (lower row) under native conditions (PBS). The two-dimensional plots are color-coded for frequency of occurrence. (Top and right histograms) Maximum projections lifetime (τ) and E_FRET. (Dashed circles) Result from two-dimensional Gaussian fits of both the 0-peak and the FRET-peak population. (Dashed lines) Centers of the fits to their position in the one-dimensional representation of E_FRET and τ data. (b) Relationship between donor fluorescence anisotropy (r) and corresponding τ. (Solid line) Expected trend according to the Perrin equation. (c) Burst-integrated fluorescence-lifetime histograms analogous to (a) for segment ε and γ under unfolding conditions (4 M GdmCl). (d) E_FRET from all experiments plotted as a function of GdmCl concentration for all mutants α to ζ. FRET efficiencies (from lifetime) in native conditions range from E_FRET = 0.34 (segment γ) to 0.59 (segment ε) and decrease during denaturation to a final value of E_FRET = 0.14 (segment γ) to 0.40 (segment ε) in 4 M GdmCl. (Solid lines) Result of monoexponential fits.